Journal
FEBS LETTERS
Volume 579, Issue 28, Pages 6365-6374Publisher
WILEY
DOI: 10.1016/j.febslet.2005.10.019
Keywords
optical biosensor; resonant waveguide grating biosensor; G protein-coupled receptor; bradykinin B-2 receptor; signaling
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The spatial and temporal targeting of proteins or protein assemblies to appropriate sites is crucial to regulate the specificity and efficiency of protein-protein interactions, thus dictating the timing and intensity of cell signaling and responses. The resultant dynamic mass redistribution could be manifested by label free optical biosensor, and lead to a novel and functional optical signature for studying cell signaling. Here we applied this technology, termed as mass redistribution cell assay technology (MRCAT), to study the signaling networks of bradykinin B-2 receptor in A431 cells. Using MRCAT, the spatial and temporal relocation of proteins and protein assemblies mediated by bradykinin was quantitatively monitored in microplate format and in live cells. The saturability to bradykinin, together with the specific and dose-dependent inhibition by a B-2 specific antagonist HOE140, suggested that the optical signature is a direct result of B-2 receptor activation. The sensitivity of the optical signature to cholesterol depletion by methyl-beta-cyclodextrin argued that B-2 receptor signaling is dependent on the integrity of lipid rafts; disruption of these microdomains hinders the B-2 signaling. Modulations of several important intracellular targets with specific inhibitors suggested that B-2 receptor activation results in signaling via at least dual pathways - G(s)- and G(q)-mediated signaling. Remarkably, the two signaling pathways counter-regulate each other. Several critical downstream targets including protein kinase C, protein kinase A, and epidermal growth factor receptor had been identified to involve in B-2 signaling. The roles of endocytosis and cytoskeleton modulation in B-2 signaling were also demonstrated. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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